Abstract The long-term goal of this project is to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER). At these locations, COPII-coated carriers are first generated, packaging cargoes for transport to ER-Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to the ER. The COPII coat is composed of two multimeric protein complexes, Sec23-Sec24 and Sec13-Sec31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII transport carrier biogenesis and anterograde transport in cells. This proposal focuses on the role of TFG, a metazoan-specific protein required for the normal trafficking of COPII- coated transport carriers. Based on our preliminary results, we hypothesize that TFG facilitates the local retention of ER-derived transport carriers, providing sufficient time for COPII coat disassembly and subsequent fusion with ERGIC membranes. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, including hereditary spastic paraplegia (HSP), suggesting a role for COPII-mediated transport in maintaining neuron function. The specific aims of this renewal application are to: 1) define mechanisms by which TFG regulates anterograde COPII-mediated cargo transport, 2) identify mechanisms by which TFG facilitates neuronal function and maintenance, and 3) determine mechanisms that underlie neurodegeneration observed in rodent models of HSP. Together, the experiments outlined in this proposal will provide fundamental new insights into how the organization of the early secretory pathway promotes the rapid anterograde transport of newly synthesized cargoes in transport carriers, which is necessary for normal human development and neuronal homeostasis.